Novel salt of fused pyrimidine compound and crystal thereof

ABSTRACT

Provided is a salt having a high selectivity to BTK and is useful as a drug ingredient for a pharmaceutical product. 
     It has been found that fumarate of Compound A is free of a characteristic of channel hydrate and is stable and excellent in absorptive property, compared to Compound A or other salts thereof.

TECHNICAL FIELD

The present invention relates to a novel salt of a compound having aninhibitory activity against Bruton's tyrosine kinase (BTK), and acrystal thereof.

BACKGROUND ART

It is known that various protein kinases exist in vivo and are involvedin the regulation of a variety of functions. Bruton's tyrosine kinase(BTK) is a protein kinase that belongs to the Tec family kinases, and isa non-receptor tyrosine kinase that plays an important role related tothe control of, for example, proliferation, survival, differentiationand activation of B-cells in the downstream of the B cell receptor (BCR)signal (Non-Patent Literature 1). An inhibitor capable of controllingthe BTK activity is considered to be useful as a therapeutic agent fordiseases associated with abnormal hyperactivity of BTK signaling pathway(for example, cancer).

Regarding a compound having BTK inhibitory activity, PCI-32765(Non-Patent Literature 2) and the compounds described in PatentLiteratures 1 and 2 are known.

The compounds disclosed in Patent Literatures 1 and 2 are also known toexhibit high inhibitory activity for EGFR (Epidermal Growth FactorReceptor) and JAK3 (Janus kinase 3) for example, in addition to BTK.However, since such a multikinase inhibitor suppresses, for example,cell proliferation by inhibiting various signaling pathways, there is aconcern about a variety of adverse effects. For example, it is knownthat EGFR binds to its ligand, for example, the epidermal growth factor(EGF), and participates in the proliferation and survival (for example,inhibition of apoptosis) of various cells (Non-Patent Literature 3).However, it is known that inhibitors targeting EGFR cause adverseeffects such as skin disorders and gastrointestinal dysfunction incommon, and it is widely supposed that these adverse effects mayberelated to the inhibition of the wild type EGFR signaling pathway(Non-Patent Literature 4).

Thus, PCI-45292 is known as a compound, which has an inhibitory activityagainst BTK with a weak inhibitory activity against EGFR (Non-PatentLiterature 5).

CITATION LIST Patent Literatures

-   Patent Literature 1: WO 2011/090760-   Patent Literature 2: WO 2009/158571

Non-Patent Literatures

-   Non-Patent Literature 1: Curr. Opin. Immunol., 2000 June; 12 (3):    282-8-   Non-Patent Literature 2: Proc. Natl. Acad. Sci. USA., 2010 Jul. 20;    107(29):13075-80-   Non-Patent Literature 3: Nature Rev. Cancer, Vol. 6, pp. 803-811    (2006)-   Non-Patent Literature 4: Nature Rev. Clin. Oncol., Vol. 6, pp.    98-109 (2012)-   Non-Patent Literature 5: American College of Rheumatology Annual    Meeting, Atlanta, Ga., 6-11 Nov. 2010)

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a salt which is a BTKinhibitor with a high selectivity, having a high inhibitory activityagainst BTK and a low inhibitory activity against other kinases such asEGFR, and which is useful as a drug ingredient for a pharmaceuticalproduct.

Solution to Problem

As a result of earnest research to solve the problem, the presentapplicant has found that(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (Compound A) represented bythe following formula (1) has a high inhibitory activity against BTK,with having a low inhibitory activity against other kinases such asEGFR, and is useful as a medicine for treating cancers, autoimmunediseases, or allergic diseases.

Then, during a research of physicochemical properties of Compound A forthe purpose of development of a formulation of Compound A, the presentapplicant has found that (1) Compound A is hard to use as a drugingredient for a pharmaceutical product, for having a characteristic asa channel hydrate of absorbing moisture in the air when a free form ofCompound A is exposed to an atmosphere of high humidity, and dischargingmoisture when exposed to an atmosphere of low humidity, (2) an acidaddition salt is produced only with tartaric acid, phosphoric acid, orfumaric acid, with regard to an acid addition salt of Compound A, and(3) even more surprisingly, among these acid addition salts, onlyfumarate is free of the characteristic of channel hydrate, and hasaccomplished the present invention.

More specifically, in an industrial production of a pharmaceuticalproduct, it is required that a drug ingredient have stability, etc.However, the stability, etc. depend on the attribute of each compound.Therefore, in a complex compound, it is difficult to predict a salthaving appropriate properties as a drug ingredient for a pharmaceuticalproduct, and accordingly, it is desired to find out, for each compound,various salts which are useful for pharmaceutical products. From such apoint of view, the present applicant has synthesized various salts ofCompound A and has researched properties, stability, etc. thereof. As aresult, our research succeeded in forming a salt of a fumarate, atartrate, a phosphate and a magnesium salt. However, among these saltsand a free form, the tartrate and the free form had the characteristicof channel hydrate and a poor solid stability; the phosphate failed tomaintain its original crystal form in a moisture absorption/desorptiontest, and in addition, had a poor solid stability; and the magnesiumsalt had a low crystal purity, for including a lot of analogoussubstances. It was found that only the fumarate could avoid thecharacteristic of channel hydrate, and at the same time, was excellentin obtaining-operability and reproducibility, and stable and excellentin absorptive property, and thus, the present invention has beenaccomplished.

That is, the present invention relates to the following 1) to 18).

1) Fumarate of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Compound A).

2)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Compound A).hemifumarate.

3)(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Compound A).monofumarate.

4) A BTK inhibitor containing the fumarate of Compound A as an activeingredient.

5) A pharmaceutical composition containing the fumarate of Compound A.

6) An anti-tumor agent or a preventive and/or therapeutic agent ofallergic diseases, autoimmune diseases or inflammatory diseasescontaining the fumarate of Compound A as an active ingredient.

7) A anti-tumor agent against a hematologic tumor or a preventive and/ortherapeutic agent of allergic rhinitis, pollinosis, atopic dermatitis,rheumatoid arthritis or systemic lupus erythematosus, which contains thefumarate of Compound A as an active ingredient.

8) Use of the fumarate of Compound A for producing a BTK inhibitor.

9) Use of the fumarate of Compound A for producing a pharmaceuticalcomposition.

10) Use of the fumarate of Compound A for producing an anti-tumor agentor a preventive and/or therapeutic agent of allergic diseases,autoimmune diseases or inflammatory diseases.

11) Use of the fumarate of Compound A for producing an anti-tumor agentagainst a hematologic tumor or a preventive and/or therapeutic agent ofallergic rhinitis, pollinosis, atopic dermatitis, rheumatoid arthritisor systemic lupus erythematosus.

12) The fumarate of Compound A for use in a BTK inhibition.

13) The fumarate of Compound A for use as a medicine.

14) The fumarate of Compound A for use in a preventive and/ortherapeutic agent of a tumor, an allergic disease, an autoimmune diseaseor an inflammatory disease.

15) The fumarate of Compound A for use in prevention or treatment of ahematologic tumor, allergic rhinitis, pollinosis, atopic dermatitis,rheumatoid arthritis or systemic lupus erythematosus.

16) A method of inhibiting BTK, containing administering an effectiveamount of the fumarate of Compound A to a subject in need thereof.

17) A method of preventing and/or treating a tumor, an allergic disease,an autoimmune disease or an inflammatory disease containingadministering an effective amount of the fumarate of Compound A to asubject in need thereof.

18) A method of treating a hematologic tumor, allergic rhinitis,pollinosis, atopic dermatitis, rheumatoid arthritis or systemic lupuserythematosus, containing administering an effective amount of thefumarate of Compound A to a subject in need thereof.

Advantageous Effects of Invention

The fumarate of Compound A of the present invention has an excellentsolid stability as a drug ingredient for a pharmaceutical product, andis capable of avoiding the characteristic of channel hydrate, comparedto Compound A or salts other than the fumarate of Compound A, andexcellent in obtaining-operability and reproducibility. Moreover, thefumarate of Compound A of the present invention exhibits an excellentoral absorption property, and is extremely useful as a pharmaceuticalproduct or as a drug ingredient for a pharmaceutical product.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates powder X-ray diffraction spectrum of monofumarate ofCompound A (amorphous) synthesized in Example 1 (the axis of ordinatesrepresents intensity (cps), and the axis of abscissas representsdiffraction angle (2θ±0.1°)).

FIG. 2 illustrates powder X-ray diffraction spectrum of hemifumarate ofCompound A (crystal) synthesized in Example 2 (the axis of ordinatesrepresents intensity (cps), and the axis of abscissas representsdiffraction angle (2θ±0.1°)).

FIG. 3 illustrates a differential scanning calorie (DSC) curve ofhemifumarate of Compound A (crystal) synthesized in Example 2.

FIG. 4 illustrates powder X-ray diffraction spectrum of monofumarate ofCompound A (crystal) synthesized in Example 3 (the axis of ordinatesrepresents intensity (cps), and the axis of abscissas representsdiffraction angle (2θ±0.1°)).

FIG. 5 illustrates a differential scanning calorie (DSC) curve ofmonofumarate of Compound A (crystal) synthesized in Example 3.

FIG. 6 illustrates a moisture absorption/desorption isothermal curve ofCompound A.

FIG. 7 illustrates a moisture absorption/desorption isothermal curve ofmonofumarate of Compound A (crystal).

FIG. 8 illustrates a moisture absorption/desorption isothermal curve ofhemifumarate of Compound A (crystal).

FIG. 9 illustrates a moisture absorption/desorption isothermal curve ofhemitartrate of Compound A.

FIG. 10 illustrates a moisture absorption/desorption isothermal curve ofmonophosphate of Compound A.

FIG. 11 illustrates an effect of monofumarate of Compound A (crystal) ina mouse collagen-induced arthritis model.

DESCRIPTION OF EMBODIMENTS

Hereinbelow, the present invention will be described in detail.

“Compound A” simply described in the present specification refers to(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamidein free form.

“Fumarate of Compound A” simply described in the present specificationmay be any one of salt forms of Compound A with fumaric acid, involvingmonofumarate and hemifumarate. The term is further used in a meaninginvolving both a crystal of fumarate of Compound A and an amorphous offumarate of Compound A. The fumarate of Compound A is preferablymonofumarate of Compound A (which may be abbreviated as “Compound Amonofumarate”) and hemifumarate of Compound A (which may be abbreviatedas “Compound A hemifumarate”); more preferably monofumarate of CompoundA (crystal), hemifumarate of Compound A (crystal) and monofumarate ofCompound A (amorphous); and particularly preferably monofumarate ofCompound A (crystal) and hemifumarate of Compound A (crystal).

In the present specification, the terms “crystal” and “amorphous” areused in usual meanings.

Multiple crystal forms which are different from one another in spatiallyorderly atomic arrangement and in physicochemical properties(polymorphisms) occur in some cases. The salt of the present inventionmay be any one of these polymorphisms, and may be a mixture of two ormore polymorphisms, or a mixture of a crystal and an amorphous.

The present invention also involves a labeled form of fumarate ofCompound A, that is, a compound having one or more atoms of Compound Aor a fumarate substituted with a radioisotopic element or anon-radioisotopic element.

In this connection, in a powder X-ray diffraction spectrum, adiffraction angle or a general pattern are important in recognizing anidentity of crystals, for a nature of data. Relative intensity of apowder X-ray diffraction spectrum can slightly vary depending ondirection of crystal growth, size of particles, or condition ofmeasurement, and therefore, should not be strictly interpreted.

A numerical value obtained from various patterns may be accompanied by aslight error due to the direction of crystal growth, size of particles,or condition of measurement thereof. Therefore, in the presentspecification, the term diffraction angle (2θ±0.1°) in the powder X-raydiffraction spectrum refers to a value which may be in a range within±0.1° of a value.

The term “in the vicinity” which is used with a peak temperature of anendothermic peak in a differential scanning calorie (DSC) curve refersto a value which approximately is the temperature, preferably refers toa value which may be within a range of ±5° C. of the value. Morepreferably, it refers to a value which may be in a range within ±2° C.of the value.

It is preferred that the monofumarate of Compound A (crystal) have thepowder X-ray diffraction spectrum as shown in FIG. 4 and/or adifferential scanning calorie (DSC) curve as shown in FIG. 5.

Here, characteristic peaks of the monofumarate of Compound A (crystal)in a powder X-ray diffraction spectrum may include 7.2°, 12.4°, 15.6°,25.9° and 27.6°, more preferably 7.2°, 12.4°, 14.4°, 15.0°, 15.6°,19.0°, 22.3°, 22.6°, 23.4°, 25.5°, 25.9° and 27.6°, in terms of thediffraction angle (2θ±0.1°).

The monofumarate of Compound A (crystal) of the present invention is acrystal having at least two or more peaks selected from the morepreferred peaks described above, preferably a crystal having at leastthree or more peaks selected from the peaks, more preferably a crystalhaving at least five or more peaks selected from the peaks, even morepreferably a crystal having at least eight or more peaks selected fromthe peaks, and particularly preferably a crystal having all of the peaksdescribed above.

The endothermic peak in a differential scanning calorie (DSC) curve ofthe monofumarate of Compound A (crystal) may include those in thevicinity of from 219° C. to 224° C., and preferably in the vicinity of223° C.

Another preferred mode of the monofumarate of Compound A (crystal) ofthe present invention may be a crystal in which a diffraction angle(2θ±0.1°) has at least two or more, preferably at least three or more,and more preferably at least five or more peaks selected from 7.2°,12.4°, 15.6°, 25.9° and 27.6° in a powder X-ray diffraction spectrum;and a peak temperature in a differential scanning calorie (DSC) curvehas an endothermic peak in the vicinity of from 219 to 224° C., andpreferably in the vicinity of 223° C. Even another preferred mode may bea crystal in which a diffraction angle (2θ±0.1°) in a powder X-raydiffraction spectrum has at least two or more, preferably at least threeor more, more preferably at least five or more, even more preferably atleast eight or more, and still more preferably all of the peaks selectedfrom 7.2°, 12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°,25.5°, 25.9° and 27.6°; and at the same time, a peak temperature in adifferential scanning calorie (DSC) curve has a endothermic peak in thevicinity of from 219° C. to 224° C., and preferably in the vicinity of223° C.

It is preferred that the hemifumarate of Compound A (crystal) have thepowder X-ray diffraction spectrum as shown in FIG. 2 and/or adifferential scanning calorie (DSC) curve as shown in FIG. 3.

Here, characteristic peaks of the hemifumarate of Compound A (crystal)in a powder X-ray diffraction spectrum may include 4.5°, 5.8°, 16.6°,20.2° and 26.4°, and more preferably 4.5°, 5.8°, 11.2°, 12.1°, 12.4°,13.4°, 16.6°, 17.3°, 18.2°, 20.2°, 26.4° and 27.1°, in terms of thediffraction angle (2θ±0.1°).

The hemifumarate of Compound A (crystal) of the present invention is acrystal having at least two or more peaks selected from the morepreferred peaks described above, preferably a crystal having at leastthree or more peaks selected from the peaks, more preferably a crystalhaving at least five or more peaks selected from the peaks, even morepreferably a crystal having at least eight or more peaks selected fromthe peaks, and particularly preferably a crystal having all of thepeaks.

The endothermic peak in a differential scanning calorie (DSC) curve ofthe hemifumarate (crystal) of Compound A may include those in thevicinity of from 197° C. to 199° C., and preferably in the vicinity of198° C.

Other preferred modes of the hemifumarate of Compound A (crystal) of thepresent invention may include a crystal in which a diffraction angle(2θ±0.1°) has at least two or more, preferably at least three or more,and more preferably at least five or more peaks selected from 4.5°,5.8°, 16.6°, 20.2° and 26.4° in a powder X-ray diffraction spectrum; anda peak temperature in a differential scanning calorie (DSC) curve has anendothermic peak in the vicinity of from 197 to 199° C., and preferablyin the vicinity of 198° C. Still other modes thereof may include acrystal in which a diffraction angle (2θ±0.1°) in a powder X-raydiffraction spectrum has at least two or more, preferably at least threeor more, more preferably at least five or more, even more preferably atleast eight or more, and still more preferably all of the peaks selectedfrom 4.5°, 5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°,26.4° and 27.1°; and at the same time, a peak temperature in thedifferential scanning calorie (DSC) curve has an endothermic peak in thevicinity of from 197° C. to 199° C., and preferably in the vicinity of198° C.

It is also possible to obtain the fumarate of Compound A of the presentinvention as an amorphous. Specifically, an amorphous of the fumarate ofCompound A of the present invention has a diffraction image exhibiting ahalo pattern which is broad and unclear in a powder X-ray diffractionspectrum, and more preferably has the powder X-ray diffraction spectrumas shown in FIG. 1.

Compound A can be synthesized, for example, according to the ReferenceExamples 1 and 2 which will be described later. The synthesis method ofCompound A is not limited to the Reference Examples 1 and 2 which willbe described later.

More specifically, methanesulfonyl chloride is reacted with(S)-N-Boc-3-pyperidinol in the presence of a tertiary amine such astriethylamine, to obtain (S)-tert butyl3-(methylsulfonyloxy)piperidine-1-carboxylate. Subsequently, thiscompound is reacted with 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-amine inthe presence of a base such as potassium carbonate, to obtain(R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate.Then, this compound is reacted with a palladium catalyst and a base inthe presence of benzo[d]oxazol-2-amine, under a carbon monoxideatmosphere, to obtain (R)-tert-butyl3-(4-amino-3-((benzo[d]oxazol-2-yl)carbamoyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-carboxylate.Subsequently, a Boc protective group is removed from this compound whichis then reacted with acryloyl chloride, to thereby obtain Compound A.

The monofumarate of Compound A of the present invention (amorphous) maybe produced, for example, in the following method.

To the Compound A is added tetrahydrofuran (THF) in an amount of from100 to 300 times, and preferably 150 times thereof, and water in anamount of from 0.01 to 1 times, preferably 0.1 times thereof. To themixture, fumaric acid in a molar amount equal to Compound A is added anddissolved.

The solvent is distilled off with azeotropically distilling the mixturewith THF several times, preferably 2 times to 5 times. Thus, it ispossible to obtain an amorphous of monofumarate of Compound A as a whitepowder.

The monofumarate of Compound A (crystal) of the present invention may beobtained as a white powder, for example, by suspending monofumarate ofCompound A (amorphous) in acetonitrile in an amount of from 5 to 50times, preferably 20 times thereof, and conducting a heating suspensionfor from 12 to 72 hours, preferably for 24 hours.

The hemifumarate of Compound A (crystal) of the present invention may beobtained as a white powder, for example, by suspending monofumarate ofCompound A (amorphous) in methyl ethyl ketone in an amount of from 10 to100 times, preferably 60 times thereof, and conducting a heatingsuspension for from 12 to 72 hours, preferably for 24 hours.

According to the fumarate of Compound A of the present invention, it ispossible to avoid the characteristic of channel hydrate of Compound A.

Generally, in a pharmaceutical product or a drug ingredient for apharmaceutical product which is prepared with a compound with thecharacteristic of channel hydrate avoided, it is known that problems instorage and in quality control in a humidity of a storage conditionthereof are reduced; and also that, when a solid preparation such as atablet or a capsule is produced, it is possible to reduce problems inpreparation due to a weight change of an active ingredient.

Therefore, it can be said that, according to the fumarate of Compound Aof the present invention, it is possible to expect a stable storage andeasy quality control, and also that, in terms of preparation, thefumarate of Compound A of the present invention is an excellent compoundwhich is easy to handle.

The fumarate of Compound A of the present invention is excellent inobtaining-operability and in reproducibility, compared to other salts ofCompound A. Specifically, for example, a salt of Compound A withhydrochloric acid, sulfuric acid, succinic acid, malic acid, citricacid, or acetic acid was not formed by the research method described inthe present specification. For example, in a formation of sodium salt ofCompound A, decomposition remarkably proceeded. When a hemi magnesiumsalt of Compound A was synthesized, the number of analogous substanceswas increased, and moreover, operation of obtaining the salt wascomplex, and the salt was hard to redissolve, for its a low solubilityto water and to an organic solvent.

The fumarate of Compound A of the present invention is easy to handle asa drug ingredient for a pharmaceutical product, and contributes toindustrial production of pharmaceutical product having a stable quality.

The fumarate of Compound A of the present invention is excellent insolid stability. It is important for a candidate compound to bedeveloped as a pharmaceutical product to have a solid stability, in anindustrial operation and in maintaining a quality. Therefore, thefumarate of Compound A of the present invention has excellent propertiesrequired for a pharmaceutical product or a drug ingredient for apharmaceutical product.

The fumarate of Compound A of the present invention is excellent in oralabsorption property, and contributes in providing an excellentpharmaceutical product with a high quality.

Among the salts of Compound A, fumarate of Compound A of the presentinvention is excellent in any of obtaining-operability, reproducibility,solid stability, and oral absorption property, while avoiding thecharacteristic of channel hydrate that Compound A has and maintaining asufficient solubility as a drug ingredient for a pharmaceutical product.

The fumarate of Compound A of the present invention has an excellent BTKinhibitory activity, and is useful as a preventive and/or therapeuticagent of, for example, cancers, tumors, and various immune diseases (forexample, allergic diseases, autoimmune diseases, and inflammatorydiseases). Furthermore, the fumarate has an excellent selectivity toBTK, and has an advantage of having reduced adverse effects derived frominhibiting other kinases (for example, EGFR) as well.

The fumarate of Compound A of the present invention has excellent BTKinhibitory activity. “BTK” according to the present specificationincludes human or non-human mammalian BTK's, and the BTK is preferablyhuman BTK. Incidentally, the term “BTK” includes its isoforms.

Furthermore, due to its excellent BTK inhibitory activity, the fumarateof Compound A of the present invention is useful as a medicine for theprevention or treatment of diseases associated with BTK. The “diseasesassociated with BTK” include diseases that undergo a decrease in theincidence rate and remission, alleviation and/or complete recovery ofsymptoms, as a result of deletion, suppression and/or inhibition of thefunctions of BTK. Examples of such diseases include cancers or tumors,allergic diseases, autoimmune diseases, inflammatory diseases,graft-versus-host disease without limitation, and are preferablycancers, tumors, allergic diseases and autoimmune diseases.

There is no particular limitation on the target cancers and tumors, andexamples thereof include epithelial cancers (for example, respiratorysystem cancers, gastrointestinal system cancers, reproductive systemcancers, and secretion system cancers), sarcomas, hematopoietic systemtumors, central nervous system tumors, and peripheral nerve tumors.Preferred examples are hematopoietic system tumors (for example,leukemia, multiple myeloma, and malignant lymphoma). Furthermore, thereis no particular limitation on the kind of the organs of tumordevelopment, and examples thereof include head and neck carcinoma,esophageal cancer, stomach cancer, colon cancer, rectal cancer, livercancer, gall bladder/bile duct cancer, biliary tract cancer, pancreaticcancer, lung cancer, breast cancer, ovarian cancer, cervical cancer,uterine cancer, kidney cancer, urinary bladder cancer, prostate cancer,testicular tumors, bone/soft tissue sarcoma, hematologic tumors,multiple myeloma, skin cancer, brain tumors, and mesothelial cancer.Preferred examples of the hematopoietic system tumors include acuteleukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia,chronic myelogenous leukemia, lymphoblastic lymphoma, myeloproliferativeneoplasms, chronic lymphocytic leukemia, small lymphocytic lymphoma,myelodysplastic syndromes, follicular lymphoma, MALT lymphoma, marginalzone lymphoma, lymphoplasmacytic lymphoma, Waldenstroemmacroglobulinemia, mantle cell lymphoma, diffuse large B-cell lymphoma,Burkitt's lymphoma, extranodal NK/T-cell lymphoma, Hodgkin's lymphoma,and multiple myeloma. Particularly preferred examples includehematologic tumors such as B-lymphoblastic leukemia/lymphoma, follicularlymphoma, mantle cell lymphoma, nodal follicular marginal zone lymphoma,diffuse large B-cell lymphoma, Burkitt's lymphoma, chronic lymphocyticleukemia, small lymphocytic lymphoma, Waldenstroem macroglobulinemia,extranodal NK/T-cell lymphoma, Hodgkin's lymphoma, myelodysplasticsyndromes, acute myelogenous leukemia, and acute lymphocytic leukemia.

There is no particular limitation on the target allergic diseases, andexamples thereof include, for example, bronchial asthma, allergicrhinitis, pollinosis, atopic dermatitis, food allergy, anaphylaxis, drugallergy, hives, and conjunctivitis. Preferred examples thereof includebronchial asthma, allergic rhinitis, pollinosis, and atopic dermatitis;and particularly preferred examples thereof include allergic rhinitis,pollinosis and atopic dermatitis.

There is no particular limitation on the target autoimmune diseases, andexamples thereof include rheumatoid arthritis, systemic lupuserythematosus, dermatosclerosis, polymyositis, Sjogren's syndrome, andBehcet's disease. Preferred examples thereof include rheumatoidarthritis and systemic lupus erythematosus, and particularly preferredexample is rheumatoid arthritis.

There is no particular limitation on the target inflammatory diseases,and examples thereof include appendicitis, blepharitis, bronchiolitis,bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, ulcerativecolitis, Crohn's disease, irritable bowel syndrome, cystitis,dacryoadenitis, contact dermatitis, dermatomyositis, cerebritis,endocarditis, endometritis, epididymitis, fasciitis, fibrositis,gastroenteritis, hepatitis, sudoriferous abscess, laryngitis, mastitis,meningitis, myelitis, myocarditis, nephritis, ovaritis, didymitis,pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonia, rectitis, prostatitis, pyelonephritis,salpingitis, nasosinusitis, stomatitis, osteoarthritis, synovitis,tendinitis, tonsillitis, uveitis, vaginitis, vasculitis, and vulvitis.Preferred examples may include ulcerative colitis, Crohn's disease,irritable bowel syndrome, contact dermatitis, cystitis, andosteoarthritis. Particularly preferred examples may include contactdermatitis, cystitis, and osteoarthritis.

When the fumarate of Compound A of the present invention is used as apharmaceutical composition, various dosage forms can be employedaccording to the purpose of prevention or treatment by incorporatingpharmaceutical carriers as necessary. The dosage form may be, forexample, any of an oral preparation, an injectable preparation, asuppository preparation, an ointment, and a patch. Any of these dosageforms can be produced by a formulation method that is publicly known andconventionally used by those skilled in the art. Particularly, a tabletfor oral administration, a coated tablet, a pill, a granularpreparation, a powder preparation, and a capsule preparation whichcontain a crystal of the fumarate of Compound A as a drug ingredient forproduction are advantageous as a stable solid preparation.

Regarding the pharmaceutical carriers, various organic or inorganiccarrier materials that are conventionally used as formulation materialsare used, and the pharmaceutical carriers are incorporated as, forexample, an excipient, a binder, a disintegrant, a lubricant, and acoating agent in solid preparations; and as a solvent, a dissolutionaid, a suspending agent, an isotonic agent, a pH adjusting agent, abuffering agent, and an analgesic agent in liquid preparations.Furthermore, if necessary, formulation additives such as an antiseptic,an antioxidant, a colorant, a flavoring/savoring agent, and a stabilizercan also be used.

Examples of the excipient include lactose, sucrose, D-mannitol, starch,crystalline cellulose, and calcium silicate.

Examples of the binder include hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, sugar powder, and hypromellose.

Examples of the disintegrant include sodium starch glycolate, carmellosecalcium, croscarmellose sodium, crospovidone, low-substitutedhydroxypropyl cellulose, and partially gelatinized starch.

Examples of the lubricant include talc, magnesium stearate, sucrosefatty acid esters, stearic acid, and sodium stearyl fumarate.

Examples of the coating agent include ethyl cellulose, aminoalkylmethacrylate copolymer RS, hypromellose, and sucrose.

Examples of the solvent include water, propylene glycol, andphysiological saline.

Examples of the dissolution aid include polyethylene glycol, ethanol,α-cyclodextrin, Macrogol400, and Polysorbate 80.

Examples of the suspending agent include carrageenan, crystallinecellulose, carmellose sodium, and polyoxyethylene hydrogenated castoroil.

Examples of the isotonic agent include sodium chloride, glycerin, andpotassium chloride.

Examples of the pH adjusting agent and the buffering agent includesodium citrate, hydrochloric acid, lactic acid, phosphoric acid, andsodium dihydrogen phosphate.

Examples of the analgesic agent include procaine hydrochloride andlidocaine.

Examples of the antiseptic agent include ethyl para-oxybenzoate, cresol,and benzalkonium chloride.

Examples of the antioxidant include sodium sulfite, ascorbic acid, andnatural vitamin E.

Examples of the colorant include titanium oxide, iron sesquioxide,Edible Blue No. 1, and copper chlorophyll.

Examples of the flavoring/savoring agent include aspartame, saccharin,sucralose, 1-menthol, and mint flavor.

Examples of the stabilizer include sodium pyrosulfite, sodium edetate,erythorbic acid, magnesium oxide, and dibutylhydroxytoluene.

In preparing an oral solid preparation, an excipient, a binder, adisintegrant, a lubricant, a colorant, and a flavoring/savoring agentare optionally added to the fumarate of Compound A, and then, forexample, a tablet, a coated tablet, a granular preparation, a powderpreparation, or a capsule preparation can be produced by a conventionalmethod.

In preparing an injectable preparation, a pH adjusting agent, abuffering agent, a stabilizer, an isotonic agent, and a local anestheticare added to the fumarate of Compound A, and subcutaneous,intramuscular, and intravenous injectable preparations can be producedby conventional methods.

The amounts of the fumarate of Compound A to be incorporated into thevarious unit dosage forms may vary depending on the symptoms of thepatient to whom this fumarate should be applied, or depending on theformulation form; however, it is generally desirable to adjust theamount to from 0.05 to 1,000 mg in an oral preparation, to from 0.01 to500 mg in an injectable preparation, and to from 1 to 1,000 mg in asuppository preparation, per unit dosage form.

Furthermore, the daily dose of a medicament having the dosage formdescribed above may vary with, for example, the symptoms, body weight,age and gender of the patient, and cannot be determinedindiscriminately. However, the dose may usually be adjusted to from 0.05to 5,000 mg, and preferably from 0.1 to 1,000 mg, per day for an adult(body weight: 50 kg), and it is preferable to administer this amountonce a day, or in divided portions in about two to three times a day.

EXAMPLES

Hereinafter, the present invention will be described more specificallyby way of Examples, but the present invention is not intended to belimited to these. Although the present invention is sufficientlydescribed by the Examples, it should be understood that various changesor modifications can be done by those skilled in the art. Therefore,such changes or modifications are involved in the present invention,unless they deviate from the scope of the present invention.

Regarding the various reagents used in the Examples, unless particularlystated otherwise, commercially available products were used. For silicagel column chromatography, a PURIF-PACK (registered trademark) SImanufactured by Schott Moritex Corp., a KP-Sil (registered trademark)Silica Prepacked Column manufactured by Biotage AB, or an HP-Sil(registered trademark) Silica Prepacked Column manufactured by BiotageAB was used. For basic silica gel column chromatography, a PURIF-PACK(registered trademark) NH manufactured by Moritex Corp., or a KP-NH(registered trademark) Prepacked Column manufactured by Biotage AB wasused. For thin layer chromatography for fractionation, a KIESELGELTM60F254, Art. 5744 manufactured by Merck KGaA, or a NH2 silica gel60F254 plate manufactured by Wako Pure Chemical Industries, Ltd. wasused. The NMR spectrum was measured with an AL400 (400 MHz; JEOL, Ltd.),a MERCURY400 (400 MHz; Agilent Technologies, Inc.) type spectrometer, oran INOVA400 (400 MHz; Agilent Technologies, Inc.) equipped with an400MNMR probe (Protasis Corp.) type spectrometer, and withtetramethylsilane as the internal reference in a case in which thedeuterated solvent contains tetramethylsilane, while in other cases,with an NMR solvent as the internal reference. All the 8 values wereexpressed in ppm.

The LCMS spectrum was measured with an ACQUITY SQD (quadrupole type)manufactured by Waters Corp. under the conditions described below.

Column: YMC-TRIART C18 manufactured by YMC Co., Ltd., 2.0×50 mm, 1.9 μm

MS detection: ESI positive

UV detection: 254 nm and 210 nm

Column flow rate: 0.5 mL/min

Mobile phase: Water/acetonitrile (0.1% formic acid)

Amount of injection: 1 μL

Gradient (Table 1)

TABLE 1 Time (min) Water Acetonitrile   0-0.1 95%  5% 0.1-2.1 95% to 5%5% to 95% 2.1-3.1  5% 95%

Furthermore, reverse phase preparative HPLC purification was carried outwith a preparative system manufactured by Waters Corp. under theconditions described below.

Column: YMC-ACTUS TRIART C18manufactured by YMC Co., Ltd., 20×50 mm, 5μm, connected with YMC-ACTUS TRIART C18 manufactured by YMC Co., Ltd.20×10 mm, 5 μm, was used.

UV detection: 254 nm

MS detection: ESI positive

Column flow rate: 25 mL/min

Mobile phase: Water/acetonitrile (0.1% formic acid)

Amount of injection: 0.1 to 0.5 mL

The meanings of abbreviations are shown below.

s: Singlet

d: Doublet

t: Triplet

q: Quartet

dd: Double doublet

dt: Double triplet

td: Triple doublet

tt: Triple triplet

ddd: Double double doublet

ddt: Double double triplet

dtd: Double triple doublet

tdd: Triplet double doublet

m: Multiplet

br: Broad

brs: Broad singlet

CDI: Carbonyldiimidazole

DMSO-d₆: Deuterated dimethyl sulfoxide

CDCl₃: Deuterated chloroform

CD₃OD: Deuterated methanol

THF: Tetrahydrofuran

DMF: N,N-dimethylformamide

DMA: N,N-dimethylacetamide

NMP: 1-Methyl-2-pyrrolidinone

DMSO: Dimethyl sulfoxide

TFA: Trifluoroacetic acid

WSC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

HOBt: 1-Hydroxybenzotriazole monohydrate

HATU:(Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaneiminiumhexafluorophosphate

DIAD: Diisopropyl azodicarboxylate

TBAF: Tetrabutylammonium fluoride

DIPEA: Diisopropylethylamine

Boc: Tert-butoxycarbonyl

Boc₂O: Di-tert-butyl dicarbonate

DMAP: Dimethylaminopyridine

Powder X-ray diffraction measurement

The powder X-ray diffraction was measured in accordance with thefollowing test conditions, after a test substance is lightly pulverizedas needed in an agate mortar.

Device: Rigaku MiniFlexII

Target: Cu

X-ray output setting: 15 mA, 30 kV

Scanning area: 2.0 to 40.0°

Step size: 0.010°

Scanning speed: 5.00° /min.

Divergence Slit: 1.25°

Scattering Slit: Open

Light receiving slit: Open

Handling of the devices including data processing was based on themethod and the process indicated in each device.

Numerical values obtained from various spectrums may slightly fluctuateaccording to direction of crystal growth, size of particles, orcondition of measurement thereof. Therefore, those numerical valuesshould not be strictly interpreted.

Thermal Analysis Measurement (Differential Scanning Calorie Measurement(DSC Measurement))

DSC measurement was measured in accordance with the following testconditions.

Device: TA Instrument Q1000

Sample: About 1 mg

Sample container: Aluminum made

Temperature rising speed: Raised by 5° C./min., up to 250° C.

Atmospheric gas: Nitrogen

Flow rate of nitrogen gas: 50 mL/min.

Handling of the devices including data processing was based on themethod and the process indicated in each device.

Reference Example 1 Synthesis of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

(Step 1) Synthesis of (S)-tert-butyl3-(methylsulfonyloxy)piperidine-1-carboxylate

20 g of (S) -N-Boc-3-piperidinol was dissolved in 100 mL of toluene, and21 mL of triethylamine and 9.2 mL of methanesulfonyl chloride were addedthereto at 0° C. The mixture was stirred for 1 hour under ice cooling,subsequently ethyl acetate and water were added thereto, and an organiclayer was separated. The organic layer was washed with a saturatedaqueous solution of sodium hydrogen carbonate, a saturated aqueoussolution of ammonium chloride and water, and then was dried overanhydrous sodium sulfate. The solvent was distilled off under reducedpressure, and thus 26.8 g of the title compound was obtained as acolorless solid.

(Step 2) Synthesis of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

A suspension solution of 14.6 g of3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine synthesized by the methoddescribed in WO 2007/126841, 25 g of (S)-tert-butyl3-(methylsulfonyloxy)piperidine-1-carboxylate obtained in Step 1, and 69g of potassium carbonate in 150 mL of DMA was heated to 100° C., and wasstirred for 10 hours. The suspension solution was cooled to roomtemperature, and then 300 mL of water was added thereto. A solid thusobtained was collected by filtration and washed with water, and thesolid was dried. Thus, 26.9 g of the title compound was obtained as ayellow solid.

Reference Example 2 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Compound A)

(Step 1) Synthesis of (R)-tert-butyl3-(4-amino-3-((benzo[d]oxazol-2-yl)carbamoyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

300 mg of (R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylateobtained in Reference Example 1 was dissolved in 3 mL of NMP. 118 mg ofbenzo[d]oxazol-2-amine, 20 mg of xantphos, and 0.15 mL ofN-methylmorpholine were added thereto, and a degassing operation wascarried out. Thereafter, 7.6 mg of palladium acetate was added thereto,and under a carbon monoxide atmosphere, the mixture was heated to 110°C. and stirred for 2 hours. After the mixture was cooled, 4.5 mL ofmethanol and 0.45 mL of a 5 N aqueous solution of sodium hydroxide wereadded thereto, and the mixture was stirred for 30 minutes at roomtemperature. Thereafter, the pH was adjusted to 5.3 with 2 N HCl, and asolid thus obtained was collected by filtration. The crude product waspurified by a silica gel column (chloroform-methanol), and thus 257 mgof the title compound was obtained as a white solid.

(Step 2) Synthesis of Compound A

5.0 g of (R)-tert-butyl3-(4-amino-3-((benzo[d]oxazol-2-yl)carbamoyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate obtained in Step 1 was suspended in 50 mL ofacetonitrile, and to the mixture was added 7.85 g of sodium iodide. 6.65mL of trimethylsilyl chloride was added dropwise thereto with stirringat room temperature, and the mixture was stirred for 1 hour. 87.5 mL ofwater and 12.5 mL of a 5 N aqueous solution of sodium hydroxide wereadded thereto, and then the system was ice-cooled. A solution preparedby dissolving 0.895 mL of acryloyl chloride to 4.1 mL of acetonitrilewas added dropwise thereto, and the mixture was stirred for 1 hour underice cooling. 50 mL of water was added thereto, and a solid thus producedwas collected by filtration, washed with water, and dried under areduced pressure. Thus, 4.13 g of the title compound was obtained as awhite solid (Compound A).

¹H-NMR (DMSO-d₆): δ ppm 1.53-1.68 (m, 1H), 1.86-1.98 (m, 1H), 2.08-2.21(m, 1H), 2.25-2.39 (m, 1H), 2.82-2.95 (m, 0.5H), 3.10-3.22 (m, 0.5H),3.23-3.37 (m, 0.5H), 3.68-3.78 (m, 0.5H), 4.04-4.14 (m, 0.5H), 4.22-4.38(m, 1H), 4.52-4.65 (m, 0.5H), 4.67-4.81 (m, 1H), 5.58-5.74 (m, 1H),6.03-6.19 (m, 1H), 6.68-6.92 (m, 1H), 7.28-7.40 (m, 2H), 7.59-7.71 (m,2H), 8.22 (brs, 2H), 8.28 (s, 1H), 12.15 (brs, 1H)

Reference Example 3 Research of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide(Compound A) and acid or base addition salts

(1) Research of Acid Addition Salts of Compound A

A research was carried out on salt formation with hydrochloric acid,sulfuric acid and phosphoric acid among inorganic acids. Specifically,Compound A was dissolved in an appropriate solvent, and each kind of theacids in an appropriate amount (from 0.5 to 1.5 equivalent) was addedthereto, and stirring was carried out for overnight, to therebydetermine whether or not a salt formation occurred. As a result, inadding hydrochloric acid or sulfuric acid to Compound A, decompositionremarkably proceeded, and therefore, the research was suspended. Inadding phosphoric acid to Compound A, monophosphate of Compound A wasobtained (Reference Example 4).

In addition, research was carried out on salt formation with fumaricacid, succinic acid, tartaric acid, malic acid, citric acid and aceticacid. Specifically, Compound A was dissolved in an appropriate solvent,and each kind of the acids in a molar amount equal to Compound A wasadded thereto, and thereafter, the solvent was distilled off to therebyprepare an amorphous of each kind of organic acids. Subsequently, thisamorphous was suspended with heating in an organic solvent (for example,methylethylketone, ethanol, ethyl acetate and butyl acetate), to therebydetermine whether or not a salt formation occurred. As a result, no saltwas formed with malic acid, citric acid or acetic acid. From anamorphous of succinic acid, although a crystalline substance wasobtained, it was not possible to obtain succinate in an amountequivalent to a theoretical amount. From fumaric acid, monofumarate ofCompound A (amorphous) was obtained, and from tartaric acid,hemitartrate of Compound A was obtained (Example 1 and Reference Example5).

(2) Research of Base Addition Salts of Compound A

A research was carried out on salt formation with sodium and magnesium,among inorganic bases. Specifically, Compound A was dissolved in anappropriate solvent, and each kind of the bases was added thereto (from0.5 to 1.5 equivalent), and stirring was carried out for overnight, tothereby determine whether or not a salt formation occurred. As a result,in salt formation with sodium, decomposition remarkably proceeded, andtherefore, the research was suspended. From the case of magnesium, hemimagnesium salt of Compound A was obtained (Reference Example 6).However, in the hemi magnesium salt of Compound A, the number ofanalogous substances was increased, and moreover, an operation ofobtaining the salt was complex, and redissolution was difficult, for itslow solubility to an organic solvent, and therefore, it was judged thatthe salt was inappropriate for a practical production.

Reference Example 4 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.monophosphate(monophosphate of Compound A)

To the Compound A obtained above (150 mg) was added THF (18 mL), andafter the mixture was heated to 70° C., was added phosphoric acid (27.3μL) thereto. Thereafter, the mixture was stirred at the same temperaturefor 72 hours, and a deposited solid was collected by filtration, thendried under a reduced pressure, and the title compound was obtained as awhite solid. Amount of yield: 158 mg, Ratio of yield: 85.9%

¹H-NMR (DMSO-d₆): δ ppm 1.51-1.69 (m, 1H), 1.87-1.97 (m, 1H), 2.09-2.21(m, 1H), 2.25-2.41 (m, 1H), 2.84-2.95 (m, 0.5H), 3.09-3.22 (m, 0.5H),3.22-3.38 (m, 0.5H), 3.67-3.83 (m, 0.5H), 4.04-4.17 (m, 0.5H), 4.23-4.40(m, 1H), 4.54-4.65 (m, 0.5H), 4.66-4.83 (m, 1H), 5.58-5.76 (m, 1H),6.05-6.22 (m, 1H), 6.70-6.96 (m, 1H), 7.32-7.48 (m, 2H), 7.61-7.75 (m,2H), 8.15-8.26 (brs, 2H), 8.30 (s, 1H)

Reference Example 5 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.hemi-L-(+)-tartrate(hemitartrate of Compound A)

The Compound A obtained above (600 mg) was dissolved in a mixture of THF(90 mL) and water (60 μL), and in the resultant solution, L-(+)-tartrate(209 mg) was poured and completely dissolved. The solvent was distilledoff by azeotropically distilling the mixture with THF twice, to therebyobtain a white solid. Methylethylketone (1.5 mL) was added to this whitesolid (130 mg) and a heating suspension was carried out at 70° C. for 21hours. The solid was collected by filtration, then dried under a reducedpressure, and obtained as a white solid. Amount of yield: 96 mg, Ratioof yield: 85.9%

¹H-NMR (DMSO-d₆): δ ppm 1.55-1.68 (m, 1H), 1.89-1.97 (m, 1H), 2.10-2.21(m, 1H), 2.25-2.40 (m, 1H), 2.85-2.95 (m, 0.5H), 3.15-3.65 (m, 1H),3.68-3.80 (m, 0.5H), 4.05-4.15 (m, 0.5H), 4.24-4.36 (m, 1H), 4.30 (s,2H), 4.53-4.62 (m, 0.5H), 4.67-4.79 (brs, 1H), 5.67 (dd, J=9.99 Hz, 1H),6.08-6.18 (m, 1H), 6.71-6.92 (m, 1H), 7.30-7.41 (m, 2H), 7.61-7.73 (m,2H), 8.22 (brs, 2H), 8.29 (s., 1H)

Reference Example 6 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.hemimagnesium salt (hemi magnesium salt of Compound A)

To the Compound A obtained above (159.8 mg) was added THF (3 mL) andmethanol (1 mL) so as to suspend. Then, 2M aqueous solution of sodiumhydroxide (185 μL) was added thereto and the Compound A was completelydissolved therein. Thereafter, magnesium chloride (17.6 mg) was pouredtherein and the mixture was stirred, to thereby obtain a white solid.After water (2 mL) was added, the solid was collected by filtration. Thesolid was washed with water (1 mL) twice, and dried under a reducedpressure to thereby obtain a white solid. Methylethylketone (1 mL) wasadded to this white solid (50 mg) and a heating suspension was carriedout at 70° C. for 24 hours. The solid was collected by filtration, thendried under a reduced pressure, and obtained as a white solid. Amount ofyield: 44.7 mg, Ratio of yield: 57.3%

¹H-NMR (DMSO-d₆) : δ ppm 1.52-1.67 (m, 1H), 1.88-2.01 (m, 1H), 2.19-2.41(m, 2H), 2.75-2.89 (m, 0.5H), 3.07-3.21 (m, ¹H), 3.57-3.71 (m, 0.5H),4.15-4.23 (m, 0.5H), 4.36-4.55 (m, 1H), 4.69-4.88 (m, 2H), 5.60-5.80 (m,1H), 6.08-6.22 (m, 1H), 6.79-6.96 (m, 1H), 7.08-7.23 (m, 2H), 7.47-7.56(m, 2H), 8.13-8.22 (brs, 2H), 8.49 (s, 1H), 10.42-10.50 (brs, 1H)

Example 1 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.monofumarate(monofumarate of Compound A (amorphous))

The Compound A obtained in Reference Example 2 (1.4 g) was dissolved inTHF (210 mL) and water (140 μL) at room temperature, and then fumaricacid (376 mg) was poured and completely dissolved therein. The solventwas distilled off with azeotropically distilling the mixture with THFtwice, to thereby obtain monofumarate of Compound A (amorphous form) asa white powder. Amount of yield: 1.57 g, Ratio of yield: 90.1%

¹H-NMR (DMSO-d₆): δ ppm 1.53-1.68 (m, 1H), 1.86-1.98 (m, 1H), 2.08-2.21(m, 1H), 2.25-2.39 (m, 1H), 2.82-2.95 (m, 0.5H), 3.10-3.22 (m, 0.5H),3.23-3.37 (m, 0.5H), 3.68-3.78 (m, 0.5H), 4.04-4.14 (m, 0.5H), 4.22-4.38(m, 1H), 4.52-4.65 (m, 0.5H), 4.67-4.81 (m, 1H), 5.58-5.74 (m, 1H),6.03-6.19 (m, 1H), 6.62 (s, 2H), 6.68-6.92 (m, 1H), 7.28-7.40 (m, 2H),7.59-7.71 (m, 2H), 8.22 (brs, 2H), 8.28 (s, 1H), 12.15 (brs, 1H)

Powder X-ray diffraction spectrum: Shown in FIG. 1.

Example 2 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.hemifumarate(hemifumarate of Compound A (crystal))

The monofumarate of Compound A obtained in Example 1 (amorphous) (450mg) was suspended in methylethylketone (27 mL), and a heating suspensionwas carried out at 80° C. for 24 hours. Through a collection byfiltration, and a subsequent drying under a reduced pressure,hemifumarate of Compound A (crystal) was obtained as a white powder.Amount of yield: 279 mg, Ratio of yield: 62.0%

¹H-NMR (DMSO-d₆): δ ppm 1.53-1.68 (m, 1H), 1.86-1.98 (m, 1H), 2.08-2.21(m, 1H), 2.25-2.39 (m, 1H), 2.82-2.95 (m, 0.5H), 3.10-3.22 (m, 0.5H),3.23-3.37 (m, 0.5H), 3.68-3.78 (m, 0.5H), 4.04-4.14 (m, 0.5H), 4.22-4.38(m, 1H), 4.52-4.65 (m, 0.5H), 4.67-4.81 (m, 1H), 5.58-5.74 (m, 1H),6.03-6.19 (m, 1H), 6.62 (s, 1H), 6.68-6.92 (m, 1H), 7.28-7.40 (m, 2H),7.59-7.71 (m, 2H), 8.22 (brs, 2H), 8.28 (s, 1H), 12.15 (brs, 1H)

Powder X-ray diffraction spectrum: Shown in FIG. 2.

Characteristic diffraction angle (2θ±0.1°):

4.5°, 5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°,26.4°, 27.1°

Differential scanning calorie (DSC) curve: Shown in FIG. 3.

Endothermic peak in Differential scanning calorie (DSC) curve: in thevicinity of from 197° C. to 199° C.

Example 3 Synthesis of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.monofumarate(monofumarate of Compound A (crystal))

The monofumarate of Compound A obtained in Example 1 (amorphous) (500mg) was suspended in acetonitrile (10 mL), and a heating suspension wascarried out at 80° C. for 24 hours. Through a collection by filtration,and a subsequent drying under a reduced pressure, monofumarate ofCompound A (crystal) was obtained as a white powder. Amount of yield:448 mg, Ratio of yield: 89.6%

¹H-NMR (DMSO-d₆): δ ppm 1.53-1.68 (m, 1H), 1.86-1.98 (m, 1H), 2.08-2.21(m, 1H), 2.25-2.39 (m, 1H), 2.82-2.95 (m, 0.5H), 3.10-3.22 (m, 0.5H),3.23-3.37 (m, 0.5H), 3.68-3.78 (m, 0.5H), 4.04-4.14 (m, 0.5H), 4.22-4.38(m, 1H), 4.52-4.65 (m, 0.5H), 4.67-4.81 (m, 1H), 5.58-5.74 (m, 1H),6.03-6.19 (m, 1H), 6.62 (s, 2H), 6.68-6.92 (m, 1H), 7.28-7.40 (m, 2H),7.59-7.71 (m, 2H), 8.22 (brs, 2H), 8.28 (s, 1H), 12.15 (brs, 1H)

Powder X-ray diffraction spectrum: Shown in FIG. 4.

Characteristic diffraction angle (2θ±0.1°):

7.2°, 12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°,25.9° and 27.6°

Differential scanning calorie (DSC) curve: Shown in FIG. 5.

Endothermic peak in Differential scanning calorie (DSC) curve: in thevicinity of from 219° C. to 224° C.

Comparative Example 1 Synthesis of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Comparative compound 1)

Synthesis was carried out in accordance with the method described in WO2008/121742 pamphlet to obtain the title compound as a white solid.

¹H-NMR (DMSO-d₆): δ ppm 1.21-1.28 (m, 1H), 1.42-1.71 (m, 1H), 1.91 (brs,1H), 2.04-2.36 (m, 2H), 2.91-3.10 (m, 1H), 3.13-3.27 (m, 1H), 3.59-3.76(m, 1H), 4.04-4.26 (m, 2H), 4.47-4.80 (m, 2H), 5.51-5.78 (m, 1H),5.96-6.21 (m, 1H), 6.64-6.95 (m, 1H), 7.14 (dd, J=11.46, 8.54 Hz, 6H),7.40-7.47 (m, 2H), 7.63-7.70 (m, 2H), 8.26 (s, 1H)

Effects of the fumarate of Compound A of the present invention wereconfirmed by the following Test Examples. In this connection, as theCompound A and the salt thereof of the present Test Examples, crystalsthereof were used, unless otherwise described.

Test Example 1 Moisture Absorption/Desorption Test

Moisture absorption/desorption test was carried out with respect toCompound A, monofumarate of Compound A, hemifumarate of Compound A,hemitartrate of Compound A and monophosphate of Compound A, to researchthe presence or absence of the characteristic of channel hydrate.

In the moisture absorption/desorption test, measurements were carriedout according to the following conditions.

A dedicated quartz holder was filled with about from 5 to 10 mg ofsample, and a weight of the sample at each humidity was measured andrecorded in a continuous manner under the following conditions. Handlingof the devices including data processing was based on the method and theprocess indicated in each device.

Device: VTI SA+ (manufactured by TA Instruments Inc.)

Drying temperature: 60° C.

Temperature rising speed: 1° C./min.

Equilibrium in drying: It is confirmed that no reduction of 0.01 wt %occurs in 5 minutes, in a range not exceeding 300 minutes.

Temperature for measurement: 25° C.

Equilibrium in humidification: It is confirmed that no increase of 0.01wt % occurs in 5 minutes, in a range not exceeding 120 minutes.

Relative humidity program: Raised by 5% RH from 5% RH to 95% RH, andlowered by 5% RH from 95% RH to 5% RH.

Charts obtained by these tests are shown in FIG. 6 to FIG. 10. Theweight changes in the range of measurement condition are shown in Table2-1 to Table 2-5.

TABLE 2-1 Result of moisture absorption/desorption test of Compound AWeight change ratio (%) Relative humidity (%) Adsorption Desorption 50.42 0.67 20 3.08 3.20 40 3.60 3.65 60 3.88 3.88 80 4.09 4.11 95 4.394.39

TABLE 2-2 Result of moisture absorption/desorption test of monofumarateof Compound A Weight change ratio (%) Relative humidity (%) AdsorptionDesorption 5 0.01 0.05 20 0.14 0.17 40 0.30 0.33 60 0.37 0.41 80 0.450.49 95 0.60 0.60

TABLE 2-3 Result of moisture absorption/desorption test of hemifumarateof Compound A Weight change ratio (%) Relative humidity (%) AdsorptionDesorption 5 0.00 0.04 20 0.07 0.11 40 0.16 0.23 60 0.28 0.40 80 0.420.54 95 0.81 0.81

TABLE 2-4 Result of moisture absorption/desorption test of hemitartrateof Compound A Weight change ratio (%) Relative humidity (%) AdsorptionDesorption 5 0.01 −0.28 20 1.25 1.17 40 1.56 1.32 60 1.67 1.45 80 1.641.72 95 3.31 3.31

TABLE 2-5 Result of moisture absorption/desorption test of monophosphateof Compound A Weight change ratio (%) Relative humidity (%) AdsorptionDesorption 5 0.01 5.76 20 0.19 8.06 40 0.36 9.29 60 0.61 10.38 80 1.2912.12 95 17.90 17.90

As shown in Table 2-1 to Table 2-5, when Compound A was humidified at arelative humidity of from 5 to 95% which is within the range of themeasurement condition, the weight change thereof was about 4.4% at amaximum. It was also confirmed that, when the humidity was lowered fromthe relative humidity of 95%, Compound A almost returned to the originalcondition. That is, it was found that Compound A had the characteristicof channel hydrate which would absorb/desorb moisture depending onhumidity.

Similarly, hemitartrate of Compound A exhibited a weight change of about3.3% at a maximum, when humidified at a relative humidity of from 5 to95%. It was also confirmed that, when the humidity was lowered from therelative humidity of 95%, Compound A almost returned to the originalcondition. That is, it was found that hemitartrate of Compound A alsohad the characteristic of channel hydrate which would absorb/desorbmoisture depending on humidity.

It was also found that, in monophosphate of Compound A, the crystal formafter the moisture absorption/desorption test did not maintain theoriginal crystal form.

In contrast, in both the monofumarate of Compound A and hemifumarate ofCompound A of the present invention, the mass change was kept at aboutless than 1% of increase, in the relative humidity of 95%, and it wasfound that the Compound A almost return to the original condition, whena humidity was lowered. Thus, it was confirmed that the fumarate ofCompound A of the present invention is capable of avoiding thecharacteristic of channel hydrate, and had superior properties as apharmaceutical product or a drug ingredient for a pharmaceuticalproduct.

Test Example 2 Solid Stability Test (Acceleration Test)

Solid stability was measured under the following conditions, withrespect to monofumarate of Compound A, hemifumarate of Compound A,hemitartrate of Compound A and monophosphate of Compound A which wereobtained in the Examples and the Reference Examples, when they werestored for 2 weeks or 4 weeks at 40° C./75% RH (sealed condition andopened condition).

Storage condition: 40° C./75% RH (Sealed and Opened) (Opened refers to acondition where a glass container has the lid removed, and is coveredwith a KimWipe).

Points of measurement: 2 weeks and 4 weeks

Storage amount: About 30 mg

Storage container: Brown glass container

Method of preparing sample solution: Sample was dissolved in 50%acetonitrile such that a concentration of the sample would be 0.4 mg/mL.

Mass of analogous substance in the sample solution was measured by HPLCanalysis. Handling of the devices including data processing was based onthe method and the process indicated in each device. (Device: ShimadzuCorporation SIL-HTc/LC-20AB)

Column: InertSustein C18, 4.6×150 mm, 3 μm, manufactured by GL SciencesInc.

MS detection: ESI positive

UV detection: 220 nm

Column temperature: 40° C.

Column flow rate: 1.0 mL/min

Mobile phase: A; 10 mmol/L phosphate buffer (pH 6.0): Acetonitrile mixedsolution (17:3), B; Acetonitrile

Amount of injection: 5μL

Gradient: Table 3

TABLE 3 Time (min) A B  0-18 100% to 90%   0% to 10% 18-30 90% to 55%10% to 45% 30-35 55% to 45% 45% to 55% 35-45  45% 55% 45-55 100%  0%

Table 4 shows the results of evaluation on the measured mass ofanalogous substance. In the Table, A, B and C refer to percentages ofthe total mass of analogous substance of less than 0.1%, 0.1% or moreand less than 0.5%, and 0.5% or more, respectively. Incidentally, thosemarked with * were measured at 2 weeks' time and the others weremeasured at 4 weeks' time.

TABLE 4 40° C./75% RH Opened Sealed monofumarate of Compound A A Ahemifumarate of Compound A A A hemitartrate of Compound A B Bmonophosphate of Compound A C* C*

As a result of this, it was found that the monofumarate of Compound Aand the hemifumarate of Compound A produced small amount of analogoussubstance, and exhibited excellent solid stabilities compared to thehemitartrate of Compound A or the monophosphate of Compound A. Thus, itwas confirmed that the fumarate of Compound A of the present inventionexhibits an excellent solid stability.

Test Example 3 Solid Stability Test (Severe Test)

Solid stability was measured under the following conditions, withrespect to monofumarate of Compound A, hemitartrate of Compound A andmonophosphate of Compound A which were obtained in the Examples and theReference Examples, when they were stored for 2 weeks or 4 weeks at 60°C.

Storage condition: 60° C. (Sealed)

Point of measurement: 2 weeks and 4 weeks

Storage amount: About 30 mg

Storage container: Brown glass container

Method of preparing sample solution: Sample was dissolved in 50%acetonitrile such that a concentration of the sample would be 0.4 mg/mL.

In the same manner as in Test Example 2, Table 5 shows the results ofevaluation on the mass of analogous substance in the sample solution,measured by HPLC analysis. In the Table, A and B refer to percentages ofthe total mass of analogous substance of less than 0.1%, and 0.1% ormore and less than 0.5%, respectively. Incidentally, the value markedwith * was measured at 2 weeks and the others were measured at 4 weeks.

TABLE 5 60° C. monofumarate of Compound A A hemitartrate of Compound A Bmonophosphate of Compound A B*

As a result, it was found that the monofumarate of Compound A producedsmall amount of analogous substance, and exhibited excellent solidstabilities compared to the hemitartrate of Compound A or themonophosphate of Compound A. Thus, it was confirmed that the fumarate ofCompound A of the present invention exhibits an excellent solidstability.

Test Example 4 Blood Concentration Measurement Test

With respect to Compound A, hemifumarate of Compound A and monofumarateof Compound A obtained in the Examples, suspensions of 50 mg/10 mL/kg interms of molecular weight of Compound A were prepared with 0.5% HPMC.These administration solutions were orally administered to mice(Balb/cA) which had been bred under a feeding condition, at a dose of 10mL per 1 kg body weight, with a sonde for oral administration. After theadministration, the mice were returned to a cage for mice and conditionswere checked. Inside the cage was provided with a condition where waterand food were accessible ad libitum. After 0.25, 0.5, 1, 2, 4 and 6hours from the administration, the mice were anesthetized withisoflurane, and 60 μL of blood was collected from orbital sinus with acapillary blood collection tube.

The collected blood was ice cooled, and blood plasma was separated by acentrifugal operation. The mice after the blood collection was returnedto an animal breeding cage, and conditions after recovery from theanesthesia were checked. When the last blood collection was finished,the mice were euthanized by cervical dislocation, after a level check ofisoflurane anesthesia.

AUC_(0-6 hr), C_(max) and T_(max) were calculated by linear-logtrapezoid method with Phoenix WinNonlin (v6.3.0) which is a softwaremanufactured by Pharsight Corporation, from a concentration of CompoundA in each blood plasma measured by MRM method with LC-MS/MS.

The results are shown in Table 6. From the test, it was found that withrespect to C_(max) (maximum concentration in blood), hemifumarate ofCompound A exhibited a value equivalent to that of Compound A, andmonofumarate of Compound A exhibited a value about two fold of that ofCompound A; and with respect to AUC_(0-6 hr) (area under the bloodconcentration-time curve, 0-6 hours from administration), hemifumarateof Compound A exhibited a value about two fold of that of Compound A,and monofumarate of Compound A exhibited a value about 1.3 times higherthan that of Compound A. Thus, it was confirmed that the fumarate ofCompound A of the present invention exhibits an excellent oralabsorptive property.

TABLE 6 Oral administration Compound monofumarate of hemifumarate ofParameter A Compound A Compound A AUC_(0-6 hr) (μM · hr) 5.45 7.33 10.32C_(max) (μM) 3.98 7.63 4.22 T_(max) (h) 0.25 0.25 0.5

Test Example 5 Measurement of BTK Inhibitory Activity (In Vitro)

In a condition set for a method of measuring inhibitory activity invitro of the compounds against BTK kinase activity, FL-Peptide 2 wasused as a substrate, since it was described in a price list of LabChip(Registered trademark) series sample consumables of PerkinElmer Co.,Ltd. that FL-Peptide 2 corresponded to a substrate peptide in ameasurement of BTK kinase activity. The refined recombinant human BTKprotein used in the test was purchased from Carna Biosciences, Inc.

With regard to the measurement of the inhibitory activity of thecompounds, firstly, monofumarate of Compound A was diluted stepwise withdimethyl sulfoxide (DMSO). Subsequently, BTK protein, a substratepeptide (final concentration was 1 μM), magnesium chloride (finalconcentration was 10 mM), ATP (final concentration was 45 μM), and aDMSO solution of the test compounds (final concentration of DMSO was 5%)were added to a buffer solution for kinase reaction (20 mM HEPES (pH7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solutionwas incubated for 40 minutes at 25° C., a kinase reaction was carriedout. The reaction was terminated by adding EDTA thereto so as to obtaina final concentration of 30 mM. Finally, a substrate peptide that wasnot phosphorylated (S) and a phosphorylated peptide (P) were separatedand detected by microchannel capillary electrophoresis with a LabChip EZReader II (PerkinElmer, Inc.). The amounts of phosphorylation reactionwere determined from the individual peak heights of S and P, and thecompound concentration at which the phosphorylation reaction could besuppressed by 50% was defined as the IC50 value (nM). The results wereshown in Table 7 below.

TABLE 7 BTK inhibitory activity IC50 value Test compound (nM)monofumarate of Compound A 1.19

From the test result, it was found that the fumarate of Compound A ofthe present invention has a BTK inhibitory activity in vitro.

Test Example 6 BTK Inhibition Selectivity Compared with EGFR KinaseInhibitory Activity (In Vitro)

1) Measurement of BTK Inhibitory Activity

The BTK inhibitory activity was measured in the same manner as in TestExample 5.

2) Measurement of EGFR Inhibitory Activity

With regard to the setting of the conditions for a method for measuringthe inhibitory activity of a compound against EGFR kinase activity invitro, it is described in the consumable reagent supplies price list forLabChip (registered trademark) series of PerkinElmer, Inc. thatFL-PEPTIDE 22 corresponds to a substrate peptide for the measurement ofEGFR kinase activity. Therefore, a biotinated peptide(biotin-EEPLYWSFPAKKK) was produced by referring to the amino acidsequence of the peptide. The purified recombinant human EGFR proteinused in the test was purchased from Carna Biosciences, Inc.

With regard to the measurement of the inhibitory activity of thecompounds, firstly, monofumarate of Compound A was diluted stepwise withdimethyl sulfoxide (DMSO). Subsequently, EGFR protein, a substratepeptide (final concentration was 250 nM), magnesium chloride (finalconcentration was 10 mM), manganese chloride (final concentration was 10mM), ATP (final concentration was 1.5 μM), and a DMSO solution of thetest compounds (final concentration of DMSO was 2.5%) were added to abuffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mMdithiotheitol, 0.01% Triton X-100), and after the solution was incubatedfor 120 minutes at 25° C., a kinase reaction was carried out. Thereaction was terminated by adding EDTA thereto so as to obtain a finalconcentration of 24 mM. Subsequently, a detection liquid containingEu-labeled anti-phosphotyrosine antibody PT66 (PerkinElmer, Inc.) andSURELIGHT APC-SA (PerkinElmer, Inc.) was added thereto, and the systemwas left to stand for 2 hours or longer at room temperature. Finally,the amount of fluorescence upon irradiation with excitation light havinga wavelength of 337 nm was measured at two wavelengths of 620 nm and 665nm, with a PHERAstar FS (BMG Labtech GmbH). The amount ofphosphorylation reaction was determined from the ratio of the amounts offluorescence at the two wavelengths, and the compound concentration atwhich the phosphorylation reaction could be suppressed by 50% wasdefined as the IC50 value (nM). 3) BTK Inhibition Selectivity

The “EGFR inhibitory activity IC50 value (nM)/BTK inhibitory activityIC50 value (nM)” was calculated based on the results obtained in theabove sections 1) and 2), and thereby the BTK inhibition selectivity ofthe test compound was identified.

TABLE 8 EGFR inhibitory activity IC50 value (nM)/BTK inhibitory activityIC50 Test compound value (nM) monofumarate of Compound A 16.90Comparative compound 1 1.3

From the test results, it was found that the BTK inhibition selectivityagainst EGFR kinase of monofumarate of Compound A of the presentinvention was about 13 times higher than that of Comparative compound 1in vitro, and monofumarate of Compound A of the present invention had anexcellent BTK inhibition selectivity. From these results, it wasdemonstrated that the fumarate of Compound A of the present inventioncould have reduced adverse effects compared with existing BTKinhibitors.

Test Example 7 Test for measuring proliferation inhibitory activityagainst cell lines expressing BTK and EGFR (in vitro), and comparison ofits selectivity

TMD8 cells, which are of a diffuse large B-cell lymphoma cell lineexpressing BTK, were suspended in RPMI1640 medium (manufactured by LifeTechnologies Corp.) containing 10% fetal bovine serum. A431 cells, whichare of an EGFR-overexpressing, highly activated human epidermoidcarcinoma cell line, were suspended in DMEM, high glucose medium(manufactured by Life Technologies Corp.) containing 10% fetal bovineserum. The cell suspensions were inoculated into each well of 384-wellflat-bottomed microplates, and the cells were cultured for 1 day at 37°C. in an incubator containing 5% carbon dioxide gas. The monofumarate ofCompound A and Comparative compound 1 were dissolved in DMSO, and thesolutions were diluted to a concentration of 500 times the finalconcentration of the test compound, using DMSO. A DMSO solution of thetest compounds was diluted with the medium used in the suspension of theeach cell, and this was added to each of the wells of the cell cultureplates such that the final concentration of DMSO would be 0.2%. Thecells were further cultured for 3 days at 37° C. in an incubatorcontaining 5% carbon dioxide gas. Counting the number of cells beforethe addition of the compounds and after the culture for 3 days in thepresence of the compounds, was carried out with a CELLTITER GLO(manufactured by Promega Corp.) on the basis of the protocol recommendedby Promega Corp. The proliferation inhibition ratio was calculated bythe following formula, and the concentration of the test compoundinhibiting 50% (GI50 (nM)) was determined.

Proliferation inhibition ratio (%)=(C−T)/(C−C0)×100

T: Luminescence intensity of a well with the test compound

C: Luminescence intensity of a well without the test compound

C0: Luminescence intensity of a well measured before the addition of thetest compound

With a comparison between the cell proliferation inhibitory activityagainst A431 cells that depends on the EGFR proliferation signaling andthe cell proliferation inhibitory activity against TMD8 cells thatdepends on the BTK proliferation signaling, it is possible to evaluatethe influence of the respective kinases at a cellular level. That is, bycalculating the “A431 cell proliferation inhibition ratio/TMD8 cellproliferation inhibition ratio”, it is contemplated that as the value ofthe ratio is larger, the selectivity to BTK over EGFR in the cellsincreases. The values of “A.431 cell proliferation inhibition ratio/TMD8cell proliferation inhibition ratio” are shown in Table 9.

TABLE 9 A431 cell proliferation inhibition ratio/ Test compound TMD8cell proliferation inhibition ratio monofumarate of Compound A 7643Comparative compound 1 117.9

From the test results, it was found that the BTK inhibition selectivityagainst EGFR kinase of monofumarate of Compound A of the presentinvention in the cell proliferation inhibition ratio in vitro was about65 times as high as that of Comparative compound 1. Thus, it was foundthat fumarate of Compound A of the present invention had an excellentBTK inhibition selectivity, not only in kinase but also in cells. Fromthe results, it was demonstrated that the fumarate of Compound A of thepresent invention could reduce adverse effects compared with existingBTK inhibitors.

Test Example 8 Mice Collagen-Induced Arthritis Model (Preventive Effect)

The test was carried out in accordance with the method described inNon-Patent Literature (Brand D D, et al., Nat Protoc. 2007; 2,1269-1275, Xu D. et al., JPET, 2012 April; 341 (1):90-103).Seven-week-old male/DBA/1 mice (CHARLES RIVER LABORATORIES JAPAN, INC.)were intracutaneously injected in the dorsum with 100 μL/body of anequal amount-mixed solution (emulsion) of 4 mg/mL of bovine type 2collagen solution (Collagen Research Center) and Complete Freund'sAdjuvant (DIFCO Inc.) (initial immunization). After 21 days therefrom,the mice were intracutaneously injected in the chine with 100 μL/body ofan equal amount-mixed solution (emulsion) of 4 mg/mL of bovine type 2collagen solution (Collagen Research Center) and Complete Freund'sAdjuvant (DIFCO) to provide an additional immunization. The oraladministration of 1 time per day was continued for 17 days setting theadministration initiation day (day 0) as the eighth day from theadditional immunization. Symptoms of arthritis in day 0, day 3, day 7,day 10, day 14 and day 17 were scored macroscopically (0: No change, 1:Swelling of a finger, 2: Swelling of two fingers or more, 3: Swelling ofinstep, 4: Swelling of all fingers extending wrist/ankle), and a totalin four limbs was obtained as a point of individual body (maximum 16points). The results are shown in FIG. 11.

FIG. 11 shows that the group administered with prednisolone (3 mg/kg)which was set as a positive control compound of the test system, wasonly in a degree of maintaining the raised arthritis score, while thegroup administered with the monofumarate of Compound A of the presentinvention (0.381 mg/kg) lowered the arthritis score effectively. Fromthe results, it was confirmed that the fumarate of Compound A of thepresent invention had an excellent therapeutic effect against rheumatoidarthritis which had already appeared.

1-27. (canceled)
 28. A method of inhibiting BTK, comprising:administering an effective amount of a fumarate of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamideto a subject in need thereof.
 29. A method of treating a tumor, anallergic disease, an autoimmune disease or an inflammatory disease,comprising: administering an effective amount of a fumarate of(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamideto a subject in need thereof.
 30. The method of claim 29, wherein thetumor is a hematologic tumor, the allergic disease is at least oneselected from the group consisting of allergic rhinitis, pollinosis, andatopic dermatitis, and the autoimmune disease is at least one selectedfrom the group consisting of rheumatoid arthritis and systemic lupuserythematosus.
 31. The method of claim 28, wherein the fumarate is(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.hemifumarate.32. The method of claim 31, wherein the fumarate comprises a crystalhaving a powder X-ray diffraction spectrum having at least two peaks atdiffraction angles (2θ±0.1°) selected from the group consisting of 4.5°,5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°, 26.4° and27.1°.
 33. The method of claim 31, wherein the fumarate comprises acrystal having a powder X-ray diffraction spectrum having at least fivepeaks at diffraction angles (2θ±0.1°) selected from the group consistingof 4.5°, 5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°,26.4° and 27.1°.
 34. The method of claim 31, wherein the fumaratecomprises a crystal having a powder X-ray diffraction spectrum havingpeaks at diffraction angles (2θ±0.1°) of 4.5°, 5.8°, 11.2°, 12.1°,12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°, 26.4° and 27.1°.
 35. Themethod of claim 31, wherein the fumarate comprises a crystal having apowder X-ray diffraction spectrum of FIG.
 2. 36. The method of claim 31,wherein the fumarate comprises a crystal having a peak temperature in adifferential scanning calorie (DSC) curve with an endothermic peak inthe vicinity of from 197° C. to 199° C.
 37. The method of claim 28,wherein the fumarate is(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.monofumarate.38. The method of claim 37, wherein the fumarate comprises a crystalhaving a powder X-ray diffraction spectrum having at least two peaks atdiffraction angles (2θ±0.1°) selected from the group consisting of 7.2°,12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°, 25.9° and27.6°.
 39. The method of claim 37, wherein the fumarate comprises acrystal having a powder X-ray diffraction spectrum having at least fivepeaks at diffraction angles (2θ±0.1°) selected from the group consistingof 7.2°, 12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°,25.9° and 27.6°.
 40. The method of claim 37, wherein the fumaratecomprises a crystal having a powder X-ray diffraction spectrum havingpeaks at diffraction angles (2θ±0.1°) of 7.2°, 12.4°, 14.4°, 15.0°,15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°, 25.9° and 27.6°.
 41. Themethod of claim 37, wherein the fumarate comprises a crystal having apowder X-ray diffraction spectrum of FIG.
 4. 42. The method of claim 37,wherein the fumarate comprises a crystal having a peak temperature in adifferential scanning calorie (DSC) curve with an endothermic peak inthe vicinity of from 219° C. to 224° C.
 43. The method of claim 37,wherein the fumarate comprises an amorphous solid exhibiting a halopattern in a powder X-ray diffraction spectrum.
 44. The method of claim37, wherein the fumarate comprises an amorphous solid having a powderX-ray diffraction spectrum of FIG.
 1. 45. The method of claim 29,wherein the fumarate is(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.hemifumarate46. The method of claim 45, wherein the fumarate comprises a crystalhaving a powder X-ray diffraction spectrum having at least two peaks atdiffraction angles (2θ±0.1°) selected from the group consisting of 4.5°,5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°, 26.4° and27.1°.
 47. The method of claim 45, wherein the fumarate comprises acrystal having a powder X-ray diffraction spectrum having at least fivepeaks at diffraction angles (2θ±0.1°) selected from the group consistingof 4.5°, 5.8°, 11.2°, 12.1°, 12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°,26.4° and 27.1°.
 48. The method of claim 45, wherein the fumaratecomprises a crystal having a powder X-ray diffraction spectrum havingpeaks at diffraction angles (2θ±0.1°) of 4.5°, 5.8°, 11.2°, 12.1°,12.4°, 13.4°, 16.6°, 17.3°, 18.2°, 20.2°, 26.4° and 27.1°.
 49. Themethod of claim 45, wherein the fumarate comprises a crystal having apowder X-ray diffraction spectrum of FIG.
 2. 50. The method of claim 45,wherein the fumarate comprises a crystal having a peak temperature in adifferential scanning calorie (DSC) curve with an endothermic peak inthe vicinity of from 197° C. to 199° C.
 51. The method of claim 29,wherein the fumarate is(R)-1-(1-acryloylpiperidin-3-yl)-4-amino-N-(benzo[d]oxazol-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide.monofumarate.52. The method of claim 51, wherein the fumarate comprises a crystalhaving a powder X-ray diffraction spectrum having at least two peaks atdiffraction angles (2θ±0.1°) selected from the group consisting of 7.2°,12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°, 25.9° and27.6°.
 53. The method of claim 51, wherein the fumarate comprises acrystal having a powder X-ray diffraction spectrum having at least fivepeaks at diffraction angles (2θ±0.1°) selected from the group consistingof 7.2°, 12.4°, 14.4°, 15.0°, 15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°,25.9° and 27.6°.
 54. The method of claim 51, wherein the fumaratecomprises a crystal having a powder X-ray diffraction spectrum havingpeaks at diffraction angles (2θ±0.1°) of 7.2°, 12.4°, 14.4°, 15.0°,15.6°, 19.0°, 22.3°, 22.6°, 23.4°, 25.5°, 25.9° and 27.6°.
 55. Themethod of claim 51, wherein the fumarate comprises a crystal having apowder X-ray diffraction spectrum of FIG.
 4. 56. The method of claim 51,wherein the fumarate comprises a crystal having a peak temperature in adifferential scanning calorie (DSC) curve with an endothermic peak inthe vicinity of from 219° C. to 224° C.
 57. The method of claim 51,wherein the fumarate comprises an amorphous solid exhibiting a halopattern in a powder X-ray diffraction spectrum.
 58. The method of claim51, wherein the fumarate comprises an amorphous solid having a powderX-ray diffraction spectrum of FIG. 1.